The flower industry is making efforts to develop new and various varieties. An effective method of producing a new variety involves changing the color of a flower, for which the traditional breeding methods have been successfully employed to produce a wide variety of colors for almost all commercial varieties. With the above methods, however, it is rare that a single species produces colored varieties coming in a wide range of different colors since a pool of genes is limited for each species.
The colors of flowers are based mainly on two types of pigments, flavonoids and carotenoids. Flavonoids contribute mainly to the colors in the range of yellow to red and blue, while carotenoids contribute to the color tones of orange or yellow. Flavonoid molecules that make a major contribution to the color of flowers are anthocyans which are glycosides of cyanidin, delphinidin, petunidin, peonidin, malvidin, and pelargonidin. Different anthocyans impart-marked changes in the color of flowers. Furthermore, the color of flowers is affected by copigmentation with colorless flavonoids, metal complex formation, glycosylation, acylation, methylation and pH of vacuoles (Forkman, Plant Breeding 106: 1, 1991).
There are a number of reports of acylated anthocyans isolated from nature including cinerarin derived from cinerarias (Senecio cruentus) (Goto et al., Tetrahedron 25: 6021, 1984), awobanin derived from dayflowers (Commelina communis) (Goto and Kondo, Angew. Chem. Int. Ed. Engl. 30: 17, 1991) and gentiodelphin derived from Gentiana Makinoi (Yoshida et al., etrahedron 48: 4313, 1992) (Monarda didyma: Kondo et al., Tetrahedron 26: 5879, 1985; perillas, pansies (Goto et al., Tetrahedron 27:2413, 1987; Wandering Jew: Idaka et al., Tetrahedron 28: 1901, 1987; Dioscorea japonica: Shoyama et al., Phytochemistry 29: 2999, 1990; red cabbage, Platycodon qrandiflorum, lobelia, delphiniums, butterfly peas: Goto and Kondo, Angew. Chem. Int. Ed. Engl. 30:17, 1991; carrots: Glabgen et al., Phytochemistry 31: 1593, 1992; morning glory: Lu et al., Phytochemistry 32: 659, 1992; Saito et al., Phytochemistry 40: 1283, 1995; Ajuga decumbens, Clinopodium gracile, Lamiums, lavender, catnip, Leonurus macranthus, Plectranthus, Prunellas, Salvias splendens Sella, Janapnese Artichoke: Saito and Harborne, Phytochemistry 31: 3009, 1992; giant water lily: Strack et al., Phytochemistry 31: 989, 1992; bellflowers: Brandt et al., 33: 209, 1993; gentians: Hosokawa et al., Phytochemistry 40: 941, 1995; hyacinth: Hosokawa et al., Phytochemistry 40: 567, 1995).
Acyl groups which modify these anthocyan-containing flavonoids are divided into two classes based on their structure: one is the aromatic acyl groups centering on hydroxy cinnamic acids, and the other is the aliphatic acyl groups such as the malonyl group. It has been observed in the experiment carried out using the anthocyanin pigment of morning glories (Pharbitis nil) that among the acyl groups transfer reactions anthocyans to which an aromatic acyl group, preferably coumaric acid or caffeic acid, is bound show a shift of the absorption maximum to the long wavelength side (Dangle et al., Phytochemistry 34: 1119, 1993).
Furthermore, for cinerarin derived from cineraria (Senecio cruentus) which has one aliphatic acyl group and three aromatic acyl groups, it has been reported that the stability of the pigment decreases in a neutral aqueous solution by removing aromatic acyl groups (Goto et al., Tetrahedron 25: 6021, 1984). For gentiodelphin derived from gentians (Gentiana makinoi) also, it has been reported that an intra-molecular stacking of the sandwich type occurs due to the presence of two aromatic acyl groups in the molecule, which results in stabilization of the pigment in an aqueous solution (Yoshida et al., Tetrahedron 48: 4313, 1992). Moreover, Yoshida et al. have demonstrated that each of glucose at position 5 and glucose at position 3′ of anthocyanin has an acyl group bound thereto (Tetrahedron 48: 4313, 1992). It has also been reported that anthocyanin in the leaves of perillas (Perilla ocimoides) is shisonin in which coumaric acid is bound to glucose at position 3 of cyanidin 3,5-diglucoside (Tetrahedron Letters 27: 2413–2416, 1978).
However, these studies have been carried out from the aspect of organic chemistry such as structural studies of natural pigments and not from the aspect of biochemistry such as efforts to isolate enzymes which transfer acyl groups.
Of the transferases which transfer acyl groups to anthocyanin pigments, there are many reports on the malonyl group transferases which transfer an aliphatic acyl, including those from a cell culture of parsley (Matern et al., Arch. Biochem. Biophys. 208: 233, 1981; Matern et al., Arch. Biochem. Biophys. 226: 206, 1983; Matern et al., Eur. J. Biochem. 133: 439, 1983), seedlings of Cicer arientium (Koster et al., Arch. Biochem. Biophys. 234: 513, 1984), and the like.
Aromatic acyl transfer reaction was first reported for Silene, a member of Caryophyllaceae (Kamsteeg et al., Biochem. Physiol. Pflanzen 175: 403, 1980), and the activity of aromatic acyltransferase has similarly been found in the soluble enzyme fraction of Matthiola (Teusch et al., Phytochemistry 26: 991, 1986).
However, these reports have been limited to a mere demonstration of the presence of enzymatic activity, and neither the corresponding enzyme proteins have been specified nor findings have been obtained on the primary structure of the enzymes much less the genes encoding them. For other aromatic acyl transferases as well no reports have elucidated the primary structure of proteins or genes. Furthermore, there are no reports of examples in which the acylating reactions of anthocyanin pigments were positively used to expand the range of colors of flowers and to grow them, or examples in which acylation was used in an attempt to stabilize anthocyanins.
On the other hand, the biochemical pathway of synthesis of anthocyanins of Petunia hybrida has been well studied (Wiering, H. and de Vlaming, P. Inheritance and biochemistry of pigments. Petunia, P49–65 (1984), Griesbach, R. J., asen, S. and Leonhardt, B. A., Phytochemistry, 30: 1729–1731, 1991), and the presence of anthocyanins which contain an acyl group is known. As the acyl group of anthocyanins of Petunia, coumaric acid or caffeic acid is known. One molecule of coumaric acid or caffeic acid is bound to rutinoside at position 3 of anthocyanin, whose chemical structure, when the anthocyanidin is malvidin, has been assigned to 3-O-(6-O-(4-O-coumaroyl)-α-D-glucopyranosyl)-5-O-β-D-gluc opyranosyl-malvidin and 3-O-(6-O-(4-O-caffeoyl)-α-D-glucopyranosyl)-5-O-β-D-gluco pyranosyl-malvidin, respectively. However, there were no reports on anthocyanins having two acyl groups.